ELISA Sample Preparation Guide

Disclaimer

This procedure should be used as a guideline only. Please keep in mind that Creative Biolabs cannot guarantee specific results for the ELISA protocol.

ELISA is a widely employed immunoassay. Typically, it is used to quantify antibodies or antigens in biological samples. Creative Biolabs summarizes the methods of sample preparation in ELISA assays, and we also provide antibody development services for different proteins, such as single domain antibodies, membrane protein antibodies, and others, for ELISA assays.

Sample Preparation Method

Cell culture supernatant

1. Pipette the cell culture medium into a centrifuge tube and centrifuge at 1,500 rpm for 10 min at 4°C.
2. Immediately aliquot the supernatant and store the samples at -80°C. Minimize freeze-thaw cycles.

Cell extracts

1. Place the tissue culture plate on ice.
2. Aspirate the medium and gently wash the cells once with ice-cold PBS.
3. Aspirate the PBS and add 0.5 mL of complete extraction buffer per 100 mm plate.
4. Scrape the cells to collect them on an inclined plate and transfer them to a pre-chilled tube.
5. Vortex briefly and incubate on ice for 15–30 minutes.
6. Centrifuge at 13,000 rpm for 10 min at 4°C to precipitate insoluble contents.
7. Aliquot the supernatant (which is the soluble cell extract) to clean the cooling tube on ice and store the sample at -80°C. Minimize freeze-thaw cycles.

Conditioning medium

1. Place the cells in a complete (serum-containing) growth medium and allow the cells to proliferate to the desired level of confluence.
2. Remove the growth medium and gently wash with a few ml of warm PBS. Repeat the washing steps.
3. Remove the last PBS wash and gently add serum-free growth medium.
4. Incubate for 1–2 days.
5. Pipette the medium into a centrifuge tube and centrifuge at 1,500 rpm for 10 minutes at 4°C.
6. Immediately aliquot the supernatant and store the sample at -80°C. Minimize freeze-thaw cycles.

Milk

1. Collect samples and centrifuge at 10,000 x g at 4°C for 2 minutes.
2. Aliquot the supernatant and store the samples at -80°C. Minimize freeze-thaw cycles.

Plasma

1. Collect whole blood into a tube containing anticoagulant or add 0.1 M sodium citrate to 1/10 of the final volume.
2. Centrifuge at 3,000 rpm for 10 minutes at 4°C.
3. Immediately aliquot the supernatant (plasma) and store the sample at -80°C. Minimize freeze-thaw cycles.

Urine

1. Collect samples and centrifuge at 10,000 x g for 2 minutes at 4°C.
2. Aliquot the supernatant and store the sample at -80°C. Minimize freeze-thaw cycles.

Saliva

1. Collect samples and centrifuge at 10,000 x g for 2 minutes at 4°C.
2. Aliquot the supernatant and store the samples at -80°C. Minimize freeze-thaw cycles.

Serum

1. Collect whole blood in untreated or anticoagulant-free tubes.
2. Incubate undisturbed at room temperature for 20 minutes.
3. Centrifuge at 3,000 rpm for 10 minutes at 4°C.
4. Immediately aliquot the supernatant (serum) and store the sample at -80°C. Minimize freeze-thaw cycles.

Tissue extracts

1. Dissect the tissue of interest with clean tools, preferably on ice, and as quickly as possible to prevent protease degradation.
2. Place the tissue in a round-bottom microcentrifuge tube and immerse it in liquid nitrogen to "fast freeze" it. Store samples at -80°C for later use or keep them on ice for immediate homogenization.
3. For ~5 mg tissue slices, add ~300 μL of complete extraction buffer (see Cell/Tissue Extraction Buffer Formulation) to the tube and homogenize with an electric homogenizer.
4. For each rinse, rinse the blade twice with 300 μL complete extraction buffer and then maintain constant agitation at 2°C for 4 hours (e.g., on an orbital shaker in a freezer).
5. Centrifuge at 13,000 rpm for 4 minutes at 20°C. Place on ice, aliquot the supernatant (which is the soluble protein extract) into freshly frozen tubes, and store samples at -80°C. Minimize freeze-thaw cycles.

NOTE: The volume of the lysis buffer must be determined based on the amount of tissue present. The typical concentration of the final protein extract is >1 mg/mL.

Cell/Tissue Extraction Buffer Formulation

1. 100 mM Tris, pH 7.4
2. 150 mM NaCl
3. 1 mM EGTA
4. 1 mM EDTA
5. 1% Triton X-100
6. 0.5% Sodium deoxycholate
7. Additional reagents required to produce the complete extraction buffer are the phosphatase inhibitor cocktail, protease inhibitor cocktail, and PMSF.

NOTE: Immediately prior to use, supplement the cell extraction buffer with a mixture of phosphatase and protease inhibitors as described by the manufacturer, and supplement PMSF to 1 mM.

Tips:

1. The recommended concentration of protein extracts is at least 1-2 mg/ml.
2. Typically, serum, plasma, cell, and tissue extracts are diluted 50% with binding buffer.
3. Centrifuge samples at 10,000 rpm for 5 minutes at 4°C to remove any precipitates before using after thawing.

All listed services and products are For Research Use Only. Do Not use in any diagnostic or therapeutic applications.